Metabolic sensors mediate hypoglycemic detection at the portal vein

The current study sought to ascertain whether portal vein glucose sensing is mediated by a metabolic fuel sensor analogous to other metabolic sensors presumed to mediate hypoglycemic detection (e.g., hypothalamic metabosensors). We examined the impact of selectively elevating portal vein concentrations of lactate, pyruvate, or beta-hydroxybutyrate (BHB) on the sympathoadrenal response to insulin-induced hypoglycemia. Male Wistar rats (n = 36), chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein (infusion), underwent hyperinsulinemic-hypoglycemic ( approximately 2.5 mmol/l) clamps with either portal or jugular vein infusions of lactate, pyruvate, or BHB. By design, arterial concentrations of glucose and the selected metabolite were matched between portal and jugular (NS). Portal vein concentrations were significantly elevated in portal versus jugular (P < 0.0001) for lactate (5.03 +/- 0.2 vs. 0.84 +/- 0.08 mmol/l), pyruvate (1.81 +/- 0.21 vs. 0.42 +/- 0.03 mmol/l), or BHB (2.02 +/- 0.1 vs. 0.16 +/- 0.03 mmol/l). Elevating portal lactate or pyruvate suppressed both the epinephrine (64% decrease; P < 0.01) and norepinephrine (75% decrease; P < 0.05) responses to hypoglycemia. In contrast, elevating portal BHB levels failed to impact epinephrine (P = 0.51) or norepinephrine (P = 0.47) levels during hypoglycemia. These findings indicate that hypoglycemic detection at the portal vein is mediated by a sensor responding to some metabolic event(s) subsequent to the uptake and oxidation of glucose.     

AE9C90CB: a novel, bladder-selective muscarinic receptor antagonist for the treatment of overactive bladder

Background and purpose:

AE9C90CB (N- [(1R, 5S, 6R)-3-azabicyclo [3.1.0] hex-6-ylmethyl]-2-hydroxy-N-methyl-2, 2-diphenylacetamide), a novel muscarinic receptor antagonist, was synthesized for the treatment of overactive bladder. Here we describe the in vitro and in vivo profiles of AE9C90CB for action in bladder over salivary gland and compare it with four agents already in clinical use (tolterodine, oxybutynin, solifenacin and darifenacin).

Experimental approach:

Radioligand binding assay and isolated tissue-based functional assay were used to evaluate affinity, potency, and receptor subtype selectivity of compounds. Inhibition of carbachol-induced increase in intravesicular pressure and salivary secretion were measured in anaesthetized rabbits to assess the functional selectivity.

Key results:

In vitro radioligand binding study using human recombinant muscarinic receptors showed that AE9C90CB had greater affinity for M₃ muscarinic receptors with pKi of 9.90 ± 0.11 and was 20-fold more selective for M₃ than for M₂ muscarinic receptors. AE9C90CB exhibited an unsurmountable antagonism on rat bladder strips (pK_B, 9.13 ± 0.12). In anaesthetized rabbits after intravenous administration, AE9C90CB dose dependently inhibited carbachol-induced increase in intravesicular pressure and salivary secretion, and exhibited functional selectivity for urinary bladder over salivary gland which was ninefold better than that of oxybutynin.

Conclusions and implications:

We have identified AE9C90CB, a compound exhibiting moderate selectivity for M₃ over M₂ receptors but greater selectivity for urinary bladder over salivary gland than oxybutynin, tolterodine, solifenacin and darifenacin. Therefore, AE9C90CB may be a promising compound for the treatment of overactive bladder with reduced potential to cause dry mouth than currently available antimuscarinic drugs.     

β-cell dysfunctional ERAD/ubiquitin/proteasome system in type 2 diabetes mediated by islet amyloid polypeptide-induced UCH-L1 deficiency

OBJECTIVE

The islet in type 2 diabetes is characterized by β-cell apoptosis, β-cell endoplasmic reticulum stress, and islet amyloid deposits derived from islet amyloid polypeptide (IAPP). Toxic oligomers of IAPP form intracellularly in β-cells in humans with type 2 diabetes, suggesting impaired clearance of misfolded proteins. In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum–associated degradation/ubiquitin/proteasome system.

RESEARCH DESIGN AND METHODS

We used pancreatic tissue from humans with and without type 2 diabetes, isolated islets from h-IAPP transgenic rats, isolated human islets, and INS 832/13 cells transduced with adenoviruses expressing either h-IAPP or a comparable expression of rodent-IAPP. Immunofluorescence and Western blotting were used to detect polyubiquitinated proteins and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) protein levels. Proteasome activity was measured in isolated rat and human islets. UCH-L1 was knocked down by small-interfering RNA in INS 832/13 cells and apoptosis was evaluated.

RESULTS

We report accumulation of polyubiquinated proteins and UCH-L1 deficiency in β-cells of humans with type 2 diabetes. These findings were reproduced by expression of oligomeric h-IAPP but not soluble rat-IAPP. Downregulation of UCH-L1 expression and activity to reproduce that caused by h-IAPP in β-cells induced endoplasmic reticulum stress leading to apoptosis.

CONCLUSIONS

Our results indicate that defective protein degradation in β-cells in type 2 diabetes can, at least in part, be attributed to misfolded h-IAPP leading to UCH-L1 deficiency, which in turn further compromises β-cell viability.Type 2 diabetes is characterized by a progressive decline in β-cell function in the face of insulin resistance. Although the mechanisms underlying β-cell dysfunction are unknown, it is likely related to the presence of β-cell endoplasmic reticulum (ER) stress (1,2), increased β-cell apoptosis (3,4), and decreased β-cell mass (3,5). The islet in type 2 diabetes is also characterized by amyloid deposits derived from islet amyloid polypeptide (IAPP) (3). Insulin resistance, the most important risk factor for development of type 2 diabetes, induces increased β-cell expression of insulin but to an even greater extent IAPP (6,7).IAPP is a 37–amino acid peptide that is coexpressed and cosecreted with insulin (8). This peptide has the propensity to form amyloid fibrils in species at risk of spontaneously developing diabetes (e.g., nonhuman primates and cats). In contrast, in rodents, IAPP is not amyloidogenic because of proline residue substitutions, and rodents do not spontaneously develop type 2 diabetes with the islet morphology present in humans (9). However, increased expression of human-IAPP (h-IAPP) in rodents may lead to type 2 diabetes with islet pathology comparable to that in humans (10–14). Intracellular IAPP toxic oligomers and ER stress have been reported in β-cells of both humans with type 2 diabetes and rodents with high expression of h-IAPP (1,15,16).The ER is responsible for synthesis, folding, and maturation of proteins. It is endowed with a quality-control system that facilitates the recognition of misfolded proteins and targets them for degradation by the ubiquitin/proteasome system (17). Efficient removal of misfolded proteins by the endoplasmic reticulum–associated degradation (ERAD) is essential to protect cells from ER stress. This is accomplished by several distinct steps. First, if a protein fails quality control, it is removed from the ER by retrograde translocation. Second, multiple ubiquitin molecules are covalently attached to the targeted protein. Third, the polyubiquinated protein is relocated to the 26S proteasome. Fourth, the ubiquitin chains are removed from the misfolded protein by a deubiquitinating enzyme, and thus it is finally rendered available for degradation by the passage through the 26S proteasome (18).We recently reported that accumulation of polyubiquitinated proteins occurs in pancreatic islets of h-IAPP transgenic mice but not in mice with comparable transgenic expression of rodent-IAPP (r-IAPP) (16). This implies that increased expression of h-IAPP may interfere with the ERAD/ubiquitin/proteasome system, and thus contribute to β-cell ER stress. β-Cells in type 2 diabetes share many characteristics of neurons in neurodegenerative diseases, such as Alzheimer''s and Parkinson''s diseases, also characterized by protein misfolding, formation of toxic oligomers of locally expressed amyloidogenic proteins, and proteotoxicity prompting ER stress (9,19). These observations imply that the mechanisms that defend against accumulation of misfolded proteins may be disrupted in β-cells in type 2 diabetes as previously documented in neurodegenerative diseases (19–23). Of interest, impairment of the ubiquitin/proteasome pathway has been shown to contribute to neurotoxicity in some neurodegenerative diseases (20–23). In particular, deficiency in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) has been observed in the brains of individuals with Alzheimer''s and Parkinson''s diseases (24). UCH-L1, a member of the deubiquinating enzyme family, is abundantly expressed in β-cells (25) and neurons (26). UCH-L1 is required to hydrolyze ubiquitin chains leading to both the release of protein targeted for degradation from the ubiquitin chain so that it can gain access to the proteasome and the generation of free monomeric ubiquitin that is then available for further cycles of the ubiquitin system. Thus, UCH-L1 deficiency impairs ubiquitin-dependent protein degradation and results in accumulation of highly ubiquitinated proteins (27–29).Given the parallels between neurons in neurodegenerative diseases and islets in type 2 diabetes, we hypothesized that the ERAD/ubiquitin/proteasome system is compromised in β-cells in type 2 diabetes. Further, we hypothesized that increased expression of amyloidogenic h-IAPP would reproduce this dysfunction. Finally, we postulated that the specific mechanism subserving the compromised ERAD/ubiquitin/proteasome system in β-cells in type 2 diabetes, and as a consequence of increased expression of h-IAPP, is decreased availability of UCH-L1.     

The relationship between urinary calcium, sodium, and potassium excretion and the role of potassium in treating idiopathic hypercalciuria

OBJECTIVES: 1) To evaluate the relationships between urinary sodium (UNa), potassium (UK), and calcium (UCa) excretion in the pediatric population; and 2) to determine the effect of increasing potassium intake in patients with idiopathic hypercalciuria and investigate whether this intervention can be offered as another mode of therapy in this patient population. DESIGN: Prospectively, we determined UNa, UK, UCa, and creatinine (Cr) concentrations in randomly collected urine samples from children on initial evaluation for urinary frequency, dysuria, hematuria, enuresis, or kidney stones to identify children with hypercalciuria. SETTING: The outpatient renal clinic of an academic hospital. PARTICIPANTS: Twenty-three black children (13 girls and 10 boys) and 77 white children (44 girls and 33 boys) 3.92 to 16.67 years of age. INTERVENTIONS: Eleven children with hypercalciuria were given potassium supplementation or placed on a high-potassium diet for at least 2 weeks. OUTCOME MEASURES: UNa to UK, UNa to Cr, UK to Cr, and UCa to Cr ratios were calculated from measured levels of urinary minerals. These were repeated in 11 hypercalciuric patients after 2 weeks of increased potassium intake. RESULTS: A total of 100 urine samples were analyzed. The UCa/Cr ratio in blacks 0.04 +/- 0.06 (mean +/- standard deviation) was significantly lower than in whites 0.16 +/- 0.12. There were 21 hypercalciuric white children versus only 1 black child. Linear regression analysis revealed a positive direct correlation between UNa/Cr and UCa/Cr in all 100 subjects and in whites alone but not in blacks. An inverse relationship existed between UK/Cr and UCa/Cr in all subjects and in whites and showed a strong trend in blacks. A marked direct relationship was found between UNa/K and UCa/Cr in all subjects (r = .43) as well as in whites (r = .59) and blacks (r = .49). One black child and 10 white hypercalciuric children were treated with "extra" K for at least 2 weeks. The UNa/K decreased from 4.73 +/- 2.28 to 1.98 +/- 1.09, and the UCa/Cr decreased from 0. 31 +/- 0.10 to 0.14 +/- 0.07, with resolution or improvement of the patients' symptoms. CONCLUSIONS: In our patient population with urinary symptoms, the UCa/Cr ratio in black children is lower and hypercalciuria less common than in white children. In both white and black populations, the UNa/K ratio had the strongest association with the UCa/Cr ratio, indicating an opposing role of UNa and UK on the UCa/Cr ratio. Increased potassium intake was found to be beneficial for hypercalciuric children by decreasing the UNa/K ratio and, consequently, the UCa/Cr ratio.     

L一粘附素单抗在缺血再灌注损伤中的作用

目的：中性粒细胞粘附在缺血再灌注损伤中有非常重要的作用。本文用ＳＤ大鼠趾长屈肌缺血再灌注损伤模型,观察Ｌ一粘附素单抗ＬＡＭ１—１１６在缺血再灌注损伤中的作用。方法：３０只ＳＤ大鼠被均分为２组：ＬＡＭ１—１１６组和生理盐水对照组。每只大鼠的一侧趾长屈肌作为正常对照,另外一侧进行 ３ ｈ缺血 ４ ｈ再灌注。结果：ＬＡＭ１— １１６组实验侧的髓过氧化物酶为正常的２倍（２．３±２．２）,生理盐水对照组则为正常的２８倍（２７．５±１１．７）（Ｐ＜０．００１）;ＬＡＭ１—１１６组的湿重比（１．１０± ０．１０）、疲劳肌力（７７． １％±１２．１％）与对照组相比（分别为 １． ２３± ０． １０和 ４９． ７％± ９ ．３％）明显改善（Ｐ＜ ０．０５）;组织学上,ＬＡＭ１—１１６组的中性粒细胞局部浸润显著减少,水肿减轻。结论：通过 Ｌ－粘附素单克隆抗体 ＬＡＭ１— １１６阻断 Ｌ－粘附素的功能,可以有效地降低中性粒细胞在再灌注肌肉中的浸润,防止组织水肿,从而改善肌肉的功能。     

水体中克雷伯菌属的分布及其在水源性急性肠道感染中的价值

俄罗斯不同气候地区不同功能水体中克雷伯菌属广泛分布。克雷伯菌属可见于遭受生物、化学污染的集中供水的地表水源,无防护的地下蓄水层,缺乏有效清洁、消毒系统的饮用水。研究表明,水体中的克雷伯菌属具有致病性和毒性,对现代药物和消毒剂(氯、紫外线)具有抗性,很容易穿透进入地下蓄水层。克雷伯菌属细菌有很强的致病性(粘附力、侵袭力、磷酸酯酶、卵磷脂酶、脱氧核糖核酸酶、溶血活性),含有致病性遗传标记cnf-1。克雷伯菌属(100 CFU/dm³)可引起急性肠道感染。在不检测总大肠菌群的情况下,检测水体尤其是饮用水中的克雷伯菌属,可以评估所用水的流行病学危险。